The cardiac transient outward current is important for repolarizing the cell membrane during the action potential. As such, factors that modulate this current can have profound effects on excitability and arrhythmogenesis. The molecular components of this current are Kv4 subunits. Early work suggested the existence of accessory subunits that modulate the expression levels and kinetics of Kv4 channels. The recently described KChIP proteins fulfill such a role. We identified another distantly related protein, frequenin (belonging to the same family of Ca2+binding proteins), expressed in human heart, which acts as an additional Kv4 accessory protein. Frequenin co-immunoprecipitates and co-localizes with Kv4 proteins, increases Kv4 membrane trafficking and slows the Kv4 inactivation process. Although initially described as expressed mainly in neurons, we find high frequenin expression levels in mouse heart. We hypothesize that KChIP2 (the prototype KChIP expressed in heart) and frequenin, are important regulators of cardiac Ito expression and function. In a multi-disciplinary approach, we will investigate this hypothesis in three Specific Aims. First, we will examine the role of KChIP2 and frequenin on Ito (and other membrane currents) in mouse cardiac myocytes by examining the Ca2+ sensitivity of Ito, as well as the effects of intervention designed to disrupt the function and expression of these proteins. Our preliminary data suggest that frequenin and KChIPs may interact. This will be investigated in Specific Aim, using co-immunoprecipitation and electrophysiological assays. In Specific Aim 3, we will investigate a proposed mechanism by which frequenin affects Kv4 currents, with the hypothesis that part of its effect on Kv4 trafficking is mediated by an interaction and stimulation of PI4Kbeta, a key regulator of the phosphoinositol pathway. We will investigate this using biochemical, trafficking, molecular biology and electrophysiological assays. The completion of these experiments will be invaluable in identifying the role of a novel accessory protein on the function of an important cardiac repolarizing K+ current. The information gained may in future be invaluable for the rational design of novel anti-arrhythmic agents.